Inhibition of Na+/K+ ATPase and Angiogenesis

All chemicals were purchased from Selleckchem (USA). The Na⁺/K⁺ ATPase inhibitor Ouabain was completely dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 10 mM. 1000 × concentrated solutions in DMSO were prepared for each assay type, except for the angiogenesis assay, where 100 × concentrated solutions were used. All assay conditions including the solvent controls were normalized to equal final concentrations of DMSO. For migration assay, the compound SKI-606 (Bosutinib) was used as reference compound. It is a very potent inhibitor of Src family kinases causing a decrease in cell motility [64 (link)]. It was demonstrated that cell proliferation was not affected at concentrations needed to inhibit cell migration [64 (link)]. Sunitinib, a multi-targeted receptor tyrosine kinase inhibitor potently targeting VEGF-R2, was used as reference compound in the angiogenesis assay. The proangiogenic ligand of VEGF-R2, human recombinant VEGF-A was provided by ProQinase GmbH (Freiburg, Germany). The viability dye Calcein-AM was purchased from Thermo Fisher Scientific (Waltham, MA, USA).